Enter the PCR template here (multiple templates are currently not supported). It is highly recommended to use refseq accession or GI (rather than the raw DNA sequence) whenever possible as this allows Primer-BLAST to better identify the template and thus perform better primer specificity checking.
A template is not required if both forward and reverse primers are entered below.
The template length is limited to 50,000 bps. If your template is longer than that, you need to use primer range to limit the length (i.e., set forward primer "From" and reverse primer "To" fields but leave forward primer "To" and reverse primer "From" fields empty).

Enter the position ranges if you want the primers to be located on the specific sites. The positions refer to the base numbers on the plus strand of your template (i.e., the "From" position should always be smaller than the "To" position for a given primer). Partial ranges are allowed. For example, if you want the PCR product to be located between position 100 and position 1000 on the template, you can set forward primer "From" to 100 and reverse primer "To" to 1000 (but leave the forward primer "To" and reverse primer "From" empty).
Note that the position range of forward primer may not overlap with that of reverse primer.

The Tm calculation is controlled by Table of thermodynamic parameters and Salt correction formula (under advanced parameters). The default Table of thermodynamic parameters is "SantaLucia 1998" and the default Salt correction formula is "SantaLucia 1998" as recommended by primer3 program.

Exon/intron selection

A refseq mRNA sequence as PCR template input is required for options in the section[?]

A refseq mRNA sequence (for example an entrez sequence record that has accession starting with NM_) allows the program to properly identify the corrsponding genomic DNA and thus find correct exon/intron boundaries.

This controls whether the primer should span an exon junction on your mRNA template. The option "Primer must span an exon-exon junction" will direct the program to return at least one primer (within a given primer pair) that spans an exon-exon junction. This is useful for limiting the amplification only to mRNA. You can also exclude such primers if you want to amplify mRNA as well as the corresponding genomic DNA.

Exon junction match

Exon at 5' sideExon at 3' side

Minimal number of bases that must anneal to exons at the 5' or 3' side of the junction
[?]

This specifies the minimal number of bases that the primer must anneal to the template at 5' side (i.e., toward start of the primer) or 3' side (i.e., toward end of the primer) of the exon-exon junction. Annealing to both exons is necessary as this ensures annealing to the exon-exon junction region but not either exon alone. Note that this option is effective only if you select "Primer must span an exon-exon junction" for "Exon junction span" option.

Intron inclusion

Primer pair must be separated by at least one intron on the corresponding genomic DNA[?]

With this option on, the program will try to find primer pairs that are separated by at least one intron on the corresponding genomic DNA using mRNA-genomic DNA alignment from NCBI. This makes it easy to distinguish between amplification from mRNA and genomic DNA as the product from the latter is longer due to presence of an intron.

With this option on, the program will search the primers against the selected database and determine whether a primer pair can generate a PCR product on any targets in the database based on their matches to the targets and their orientations. The program will return, if possible, only primer pairs that do not generate a valid PCR product on unintended sequences and are therefore specific to the intended template. Note that the specificity is checked not only for the forward-reverse primer pair, but also for forward-forward as well as reverse-reverse primer pairs.

Primer-blast tries to find target-specific primers by placing candidate primers on unique template regions that are not similar to other targets. However, in some cases, primer-blast cannot determine if a database sequence is an intended target or not, thus the user guidance might be helpful (For example, when your template is a polymorphic form or a partial region of an entry in the search database, or when the database such as the nr contains redundant entries of your template).
The "Automatic" option will ask for user guidance only when the program does not find sufficient unique template regions while the "User guided" option will always ask for user guidance if your template shows high similarity to any other database sequences.

Refseq representative genomes:
&nbsp&nbsp&nbsp This database contains NCBI RefSeq Reference and Representative genomes across broad taxonomy groups including eukaryotes, bacteria, archaea, viruses and viroids. These genomes are among the best quality genomes available at NCBI. This database contains minimum redundancy in genome representation. For the eukaryotes, only one genome is included per species (However, alternate loci of eukaryotic genomes are included where applicable). For other species, genomes from diverse isolates of the same species may be included. Mitochondrion genomes are included where applicable.

Genomes for selected organisms (primary reference assembly only):
&nbsp&nbsp&nbspThese are complete or nearly complete genome sequences from primary chromosome assemblies (i.e., no mitochondrion or alternate loci) for selected organisms including apis mellifera, bos taurus, danio rerio, dog, drosophila melanogaster, gallus gallus, human, mouse, pan troglodytes and rat. Although sequences in this database are completely covered by the Refseq representative genomes database, it does not contain the alternate loci and therefore has even less redundancy than the Refseq representative genomes database. This database is recommended if you are not concerned about missing alternate loci or mitochondrion sequences.

Custom:
&nbsp&nbsp&nbspYou can use your own sequences (accession number, gi, or FASTA sequence) as a search database.

You can choose to exclude sequences in the selected database from specificity checking if you are not concerned about these. There are a large of number of predicted Refseq transcripts in the Refseq mRNA, Refseq RNA and nr database. There are also many uncultured/environmental sample sequencesare in the nr database.

Organism

Enter an organism name (or organism group name such as enterobacteriaceae, rodents), taxonomy id or select from the suggestion list as you type.
[?]

This will limit the primer specificity checking to the specified organism. It is strongly recommended that you always specify the organism if you are amplifying DNA from a specific organism (because searching all organisms will be much slower and off-target priming from other organisms is irrelevant). Click on "Add more organisms" label if you want to restrict to multiple organisms (enter only one organism in each input box).

You can use a regular entrez query to limit the database search for primer specificity. For example, enter a GenBank accession number to limit search to that particular sequence only (Caution: this means the primer specificity will NOT be checked against any other sequences except the specified one).

Primer specificity stringency

Primer must have at least
total mismatches
to unintended targets, including
at least
mismatches within the last
bps at the 3' end.
[?]

This requires at least one primer (for a given primer pair) to have the specified number of mismatches to unintended targets. The larger the mismatches (especially those toward 3' end) are between primers and the unintended targets, the more specific the primer pair is to your template (i.e., it will be more difficult to anneal to unintended targets). However, specifying a larger mismatch value may make it more difficult to find such specific primers. Try to lower the mismatch value in such case.

This is another parameter that can be used to adjust primer specificity stringecy. If the total number of mismatches between target and at least one primer (for a given primer pair) is equal to or more than the specified number (regardless of the mismatch locations), then any such targets will be ignored for primer specificity check. For examaple, if you are only interested in targets that perfectly match the primers, you can set the value to 1. You can also lower the E value (see advanced parameters) in such case to speed up the search as the high default E value is not necessary for detecting targets with few mismatches to primers.
Additionally this program has limit detecting targets that are too different from the primers...it will detect targets that have up to 35% mismatches to the primer sequences (i.e., a total of 7 mismatches for a 20-mer).
You may need to choose more sensitive blast parameters (under advance parameters) if you want to detect targets with a higher number of mismatches than default.

If enabled, this program will NOT exclude the primer pairs that can amplify one or more mRNA splice variants from the same gene as your PCR template, thus making primers gene-specific rather than transcript-specific (Note that it is NOT intended to generate primers that will amplify all variants. It only means that the primers may amplify one or more other slice variants, in addition to the one you have specified). Enabling this option will make it much easier to find gene-specific primers since there is no need to distinguish between splice variants. This option requires you to enter a refseq mRNA accession or gi or fasta sequence as PCR template input because other type of input may not allow the program to properly interpret the result.

Maximum number of target sequences (with unique sequence identifier) blast will generate for primer-blast to screen for primer pair specificities. Note that the actual number of similarity regions (or number of hits) may be much larger than this (for example, there may be a large number of hits on a single target sequence such as a chromosome). Choose a higher value if you need to perform more stringent search.

Expected number of chance matches in a random model. A higher E value should be used if you want more stringent specificity checking (i.e., to identify targets that have more mismatches to the primers, in addition to the perfectly matched targets). On the other hand, a lower E value is recommended if you are only interested in perfect or nearly perfect matches as this will significatly shorten the search time.

The minimal number of contiguous nucleotide base matches between the query sequence and the target sequence that is needed for BLAST to detect the targets. Set a lower value if you need to find target sequences with more mismatches to your primers. However this will increase the search time.

The maximum number of candidate primer pairs to screen in order to find specific primer pairs (The candidate primers are generated by primer3 program). Increasing this number can increase the chance of finding a specific primer pair but the process will take longer.

The maximum number of Gs or Cs allowed in the last five 3' bases of a left or right primer.

Secondary Structure Alignment Methods

Use Thermodynamic Oligo AlignmentUse Thermodynamic Template Alignment (warning: search may be very slow with this option on)[?]

The option "Use Thermodynamic Oligo Alignment" instructs Primer3 to use thermodynamic alignment models (instead of old traditional secondary structure alignment) for calculating the propensity of oligos to form hairpins and dimers while the option "Use Thermodynamic Template Alignment" instructs Primer3 to use thermodynamic alignment models (instead of old traditional secondary structure alignment) for calculating the propensity of oligos to anneal to undesired sites in the template sequence.

E.g. 401,7 68,3 forbids selection of primers in the 7 bases starting at 401 and the 3 bases at 68. Or mark the source sequence with < and >: e.g. ...ATCT<CCCC>TCAT... forbids primers in the central CCCC.

Enter a list of space separated nucleotide positions. This requires that the left or the right primers to span a junction that is just 3' of any such positions. For example, entering "50 100" would mean that the left or the right primers must span the junction between nucleotide position 50 and 51 or the junction between position 100 and 101 (counting from 5' to 3'). You can also specify in the fields below the minimal number of nucleotides that the left or the right primer must have on either side of the junctions. This option is useful if you want a primer to a span specific junction on the template. Note that this option cannot be used in association with the "Exon/intron selection" options above.

5' side overlaps3' side overlaps

Minimal number of nucleotides that the left or the right primer must have at the 5' or 3' side of the junctions

The millimolar concentration of divalent salt cations (usually MgCl2+ in the PCR). Primer3 converts concentration of divalent cations to concentration of monovalent cations using formula suggested in the paper Ahsen et al., 2001.
[Monovalent cations] = [Monovalent cations] + 120*(v([divalent cations] - [dNTP])). According to the formula concentration of desoxynucleotide triphosphate [dNTP] must be smaller than concentration of divalent cations. The concentration of dNTPs is included to the formula beacause of some magnesium is bound by the dNTP. Attained concentration of monovalent cations is used to calculate oligo/primer melting temperature. See Concentration of dNTPs to specify the concentration of dNTPs.

The nanomolar concentration of annealing oligos in the PCR. Note that this is not the concentration of oligos in the reaction mix but of those annealing to template. Primer3 uses this argument to calculate oligo melting temperatures. The default (50nM) works well with the standard protocol used at the Whitehead/MIT Center for Genome Research--0.5 microliters of 20 micromolar concentration for each primer oligo in a 20 microliter reaction with 10 nanograms template, 0.025 units/microliter Taq polymerase in 0.1 mM each dNTP, 1.5mM MgCl2, 50mM KCl, 10mM Tris-HCL (pH 9.3) using 35 cycles with an annealing temperature of 56 degrees Celsius. This parameter corresponds to 'c' in Rychlik, Spencer and Rhoads' equation (ii) (Nucleic Acids Research, vol 18, num 21) where a suitable value (for a lower initial concentration of template) is "empirically determined". The value of this parameter is less than the actual concentration of oligos in the reaction because it is the concentration of annealing oligos, which in turn depends on the amount of template (including PCR product) in a given cycle. This concentration increases a great deal during a PCR; fortunately PCR seems quite robust for a variety of oligo melting temperatures.

With this option on, the program will automatically retrieve the SNP information contained in template (using GenBank accession or GI as template is required) and avoid choosing primers within the SNP regions.

If the default "Automatic" setting is selected, the program will automatically select the repeat database using the following rules.

1. If a repeat database is available from the same organism as specified in the "Organism" field by user (see above), then that repeat database will be used. For example, if "Human" is specified, then the human repeat database will be selected.

2. If a repeat database from the same organism is not available, the database from the closest parent of that organism in the taxonomy tree will be selected. For example, the rodent repeat database will be selected if "Mouse" is specified in "Organism" field. However, no repeat database will be selected if "Gallus gallus" is specified since a repeat database from its taxonomical parents is not available.